9th IJSO, Tehran, I.R.of Iran
December 5th, 2012
Name: ------
Seat Number:------
Practical Examination
Physics, Chemistry, Biology
Complete the following:
STUDENTS' NAMES:GROUP NO.:
COUNTRY:
SIGNATURES:
Theoretical Competition, Answer Sheet
9th IJSO, Tehran, I.R.of Iran
December 5th, 2012
Name: ------
Seat Number:------
The purpose of this experiment from germinated wheat.
Then to determine the size of these DNA fragments by gel electrophoresis.
DNA extraction from sprouted wheat
Note: More than one of choices may be correct, and you need to mark all corrected answers to get complete scores.Strike a tick on the correct answer.
A part of DNA extraction procedure (A to E) has been already done by laboratory staff.
DNA can be isolated from a number of plant tissues. The sprouted wheat is an excellent source of DNA.
This part of the procedure has been done for you.
- 2 grams of dried powder of sprouted wheat was placed in tube.
- A solution containing buffers, salt and detergents was added to the tissue.
- The tissue was incubated at 60oC for 20 minutes with frequent mixing. Tubes containing the sprouted wheat mixture were transferred to an ice bath for a few minutes to quickly cool it to approximately 25°C. Swirled gently during this period.
- Tubes were placed on the table.
Students should start their experiments from A stage.
We have made a solution containing DNA and give 3 mL of it to all groups in a test tube.
A-Tilt your test tube and slowly pour alcohol (Ethanol) into the tube, down the side so that it forms a layer on top of the wheat extract. Pour until you have about the same amount of alcohol in the tube as wheat mixture.
B-During about a 5-minute period, observe any change that may appear. After that, you should be able to answer the questions 1 & 2.
1- What change(s) do you observe after adding alcohol to the DNA solution ?
- Two separate phases are observed.
- Appearance of milky whiteclumps after 5 minutes.
- DNA clumps are made in the solution but they are invisible.
- Appearance of a milky white homogeneous solution.
C-Use a yellow tip or metal loop to draw up the DNA clump.
D-Transfer the collected DNA into "Your Sample" tube containing dye solution.
The final step requires the cold alcohol. The solubilized DNA contacts the alcohol where thetwo liquid layers meet. The alcohol dehydrates and precipitates the DNA, as DNA is insolublein the alcohol (especially cold alcohol).
2-What happens during the 5-minute period after adding ethanol to the test tube?
I.DNA clumps appear immediately after the addition of ethanol.
II.A very thin string of DNA appears after the addition of ethanoland its amount gradually increases.
III.DNA clumpsseparate from the lower phase and theyfloaton the upper phase.
IV.DNA clumps sink to the bottom.
What is Electrophoresis?
Electrophoresis is a technique used to separate a mixture of macromolecules such as DNA and RNA fragments or proteins by their physical properties.DNA molecules which are to be analyzed are set upon a viscous medium, the agarose gel, and are induced to migrate through the gel.
Agarose is a natural polymer made from seaweed and it makes a porous network when it is mixed with water to make the gel.
Bromophenol Blue and Xylene Cyanol dyes migrate through agarose gel at roughly the same rate as double-stranded DNA fragments of 500 and 4000 base pairs, respectively.
3-What induces DNA molecules to migrate through the agarose gel?
I.External electric field
II.Gravitational field
III.Electrical force between molecules
IV.Induced magnetic field
V.Hydrophobic Interactions
4-In the experiment, the main factorslowing down themigration rate of the DNA molecules inside the agarose gel is ...
I.Electric charge of DNA molecule
II.Electric field in the gel
III.Size of the DNA molecule
IV.Thickness of the gel
5- DNA molecule has a …………… and migrates to the ………...
I.Negative electric charge -anode
II.Positive electric charge - anode
III.Positive electric charge - cathode
IV.Negative electric charge - cathode
6-Which of the following options complete the phrase below correctly?
The more the concentration of the agarose in the gel, the ……….. of the gel.
I. smaller the pores
II.bigger the pores
For each group an agarose gel is provided. Each agarose gel contains 7 wells in which the samples will be loaded. For loading your sample in the wells, take a syringe, join it to the yellow tip and draw the sample. The samples in the yellow tip should be less thanhalf of the length of the tips. Load in the well only one drop of the sample. For loading each well sample, use a new tip. You should be aware not to destroy the well. if you cannot do the procedure , ask the lab assistant for help but you will be penalized ...... mark(s).
The wells should be allocated to the samples as follows:
- 1 & 2:your sample, which is marked by a smiley face. Number 1 is for practice of loading and number 2 is for the actual run.
- 3, leave it empty.
- 4, sample X1
- 5, sample X2
- 6, sample X3
DNA Electrophoresis Apparatus
- Measure the voltage of the power supply and report it.
In this part first set up the test and then asks for assistant to check the system.
- Measure the current in the circuit and the distance travelled by the molecules in the tank. Record your data in the table. The voltage of the powersupply is assumed to be stable. Measure for4 samples at the same time. Start at t=0 andmeasuretill t=40 minutes;use time intervals of 5 minutes. Sample X2 contains 2 different kinds of molecules called p and q. Moleculep appears as light blue color belt and molecule q appears as dark blue color belt which appear when you run the samplesusing electrophoresis.
- Calculate the resistance and record in the table.
Time
in min. / Current(I)
in mA / Distance (x)
Inmm / Resistance (R)
InΩ
Your samples / X1 / X2 / X3
p / q
0
5
10
15
20
25
30
35
40
- Plot a graph of electric current versus the time.
- Plot a graph of electrical resistance versus the time.
- For X1 and X3 samples, Plot a graphof distance versus the time. Label your graphs by suitable labels.
- Supposing that the electric field within the tank is uniform. Estimate its value.
Molecules move with terminal speed in the gel when the imposed force on molecules is equal to friction force inside the gel.Friction force is equal to friction coefficient multiplied terminal speed.
- Assume the electric charge of each molecule is equal to 110-16 C. For X1 and X3 samples,calculate the value of friction coefficient of gel.
- Mobility by definition is ratio of terminal speed of molecules to imposed electric field. For X1 and X3 samples, Calculate the mobility.
During the electrophoresis, it is observed that bubbles are continuously evolved from the electrodes. Write down the chemical formula of the gases evolved at,
Anode:
Cathode:
Write down the electrochemical half-reactions which are taking place at
Anode:
Cathode:
Use suitable arrows ( for increase, for decrease or for no change) to fill in the blanks :
The pH of the solution near anode during the electrophoresis:
The pH of the solution near cathode during the electrophoresis:
The pH of the total solution after the electrophoresis:
- Restriction enzymes cut DNA molecules atdesignated points and so produce DNA fragments. A DNA sample was treated with a restriction enzyme at the points shown by arrows.The DNA fragments were then separated using electrophoresis. Which of the schemes below(1 to 5) is the most probable result ? Circle the correct answer.
1 / 2 / 3 / 4 / 5
If this DNA molecule was circular which pattern would be the correct answer after complete digestion? Circle the correct answer.
1 / 2 / 3 / 4 / 5